Tuberculosis (TB), caused by the bacterium, Mycobacterium tuberculosis, has re-emerged as a major infectious disease worldwide and an estimated 2 million people died from the disease annually. For example, in Hong Kong alone, it has in recent years accounted for the greatest number of cases of all noticeable infectious diseases, totaling about 7,000 cases a year. The first-line anti-tuberculosis drug regimen, consisting of rifampin, isoniazid, pyrazinamide, ethambutol and streptomycin, is generally effective if adhered to as prescribed. However, the antibiotic treatment usually lasts for 6 to 8 months and the unpleasant side-effects can lead to non-compliance to the prescribed regimen, which may in turn result in the selection of resistant organisms and therefore treatment failure. This partly contributes to the emergence of multidrug-resistant strains of M. tuberculosis (MDR-MTB). Since early 1990s, outbreaks of MDR-MTB have been reported with increasing frequency, especially among human immuno-deficiency virus-positive patients in the United States and Europe. It has been shown that MDR-MTB are genetically diverse, which suggest that they may be unrelated to each other and had probably evolved independently (Z. Fang et al., J. Clin. Microbiol. 1999; 37:998-1003, PMID 10074516). Recent surveys in Hong Kong indicated multidrug resistance in 0.2 to 0.6% of MTB isolates.
As outbreaks of MDR-MTB often lead to high mortality, the need for rapid identification of drug resistance has increased. Furthermore, when a diagnosis of TB is confirmed, early initiation of chemotherapy is desirable in order to prevent complications in the patient and transmission of TB to others. Drug susceptibility testing is required by clinicians to choose the most effective anti-tuberculosis agents and to evaluate patient's response to chemotherapy. However, many current susceptibility tests, which usually involve culturing the slow-growing mycobacteria in the presence of antibiotics, take a rather long period of 5 to 14 days. Various other tests have been available, some of them are rather complicated and involve sequencing a portion of the gene of a sample. Kapur, V. et al. (J. Clin. Mocrobiol. 1999; April; 32(4):1095-8, PMID No. 8027320) have used automated DNA sequencing to characterize mutations associated with rifampin resistance in a 69-bp region of the gene, rpoB, encoding the beta subunit RNA polymerase in M. tuberculosis. U.S. Pat. No. 5,643,723 discloses a detection of a genetic locus encoding resistance to rifampin in Mycobacterial cultures. This detection method also comprises a sequencing step to determine the presence or absence of M. tuberculosis or mutants. U.S. Pat. No. 5,789,173 discloses a method for anti-microbial susceptibility testing to screen antibiotics. This method does provide a more rapid anti-microbial susceptibility testing to screen different antibiotics. U.S. Pat. No. 5,851,763 discloses a detection method of antibiotic resistance in M. tuberculosis. In this prior art, DNA sequences from M. tuberculosis are analyzed by Southern blotting and hybridization. In particular, DNA fragments can be separated on agarose gels and denatured in situ. The fragments can then be transferred from the gel to a water insoluable solid, porous support or an activated cellulose paper.
DNA mutations in the genes conferring resistance to anti-tuberculosis drugs have been identified, and include thirty-two base substitutions, four deletions and two insertion mutations have been detected in a central segment of the rpoB gene. This is shown in FIGS. 1 and 2. These mutations are believed to confer resistance to rifampin by decreasing the binding affinity of rifampin to the β-subunit of RNA polymerase. Rifampin inhibits transcription upon binding to the bacterial RNA polymerase. A method disclosed by Telenti A., et al. (Antimicrob Agents Chemother 1993 October; 37(10):2054-8, PMID No. 8257122) were able to detect all seventeen different rifampin known mutations at that time.
The present invention provides a method which can rapidly detect at least some known mutations in the RNA polymerase beta subunit (rpoB) gene. For instance, the method according to the present invention allows detection of the mutations in as short as 1 to 2 days which is significantly shorter than 5 to 14 days for using antibiotics susceptibility test with which culturing slow growing bacteria is needed. Further, the method is easy to perform and is therefore desirably cost effective to be performed on a large-scale basis. The results produced therefrom are also reliable and readily detectable. This is due to the use of both control and test arrays to compete for the same amplification products. In an unlikely event that the control and test arrays produce contradictory results, it can easily be detected and minimal mis-diagnosis results. Finally, it is envisaged that the present invention is easily adapted to automation.